Pharmaceutical compositions comprising an integrin alpha4 antagonist for use in treating ocular inflammatory conditions

ABSTRACT

The invention relates to antagonists of integrin α4 and their use in pharmaceutical compositions, primarily topically administered ophthalmic compositions. The pharmaceutical compositions are useful for treating ocular inflammatory conditions, such as dry eye disease, non-infectious uveitis (e.g., anterior, intermediate, posterior, pan-uveitis), non-infectious conjunctivitis, iritis, or scleritis in animals, and particularly mammals, including humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/742,867, having an international filing date of Jul. 7,2016, which is a U.S. National Phase Application of InternationalApplication No. PCT/IB2016/054073, filed Jul. 7, 2016, which claimspriority to U.S. Provisional Patent Application No. 62/189,813, filedJul. 8, 2015, the entire text of which is hereby incorporated byreference into this patent application.

FIELD OF THE INVENTION

This invention relates a pharmaceutical composition, such as a topicalophthalmic composition, comprising an integrin α4 antagonist for use intreating ocular inflammatory conditions, including dry eye disease. Thisinvention also relates to a method for treating ocular inflammatoryconditions, including dry eye disease, in humans and other mammals byadministering a pharmaceutical composition, such as a topical ophthalmiccomposition, comprising an integrin α4 antagonist.

BACKGROUND OF THE INVENTION

Dry eye disease (DED) is one of the most common and discomforting eyedisorders. It has been defined as a multifactorial ocular surfacedisease more prevalent in women and the elderly. DED is associated withsymptoms of discomfort, visual disturbance, tear film instability andinflammation of the ocular surface leading to potential damage to theocular surface tissues (Research in Dry Eye DEWS Report 2007). Thepro-inflammatory milieu is characterized by increased levels ofcytokines and chemokines in the tear film, cornea and conjunctiva, andincreased autoreactive T-cell infiltration of the conjunctivalepithelium and sometimes lacrimal gland (Pflugfelder et al., 1999; dePaiva et al., 2009a; Massingale et al., 2009); as reviewed by Stern andcolleagues (Stern et al., 2010; Stern et al., 2013). Alteration of thetear film composition (mucins, lipids, proteins) and decreased volumelead to tear film abnormalities that contribute to the disease cycle.

Subjecting mice to a controlled environment of desiccating stress (OS)results in ocular surface pathology reminiscent of human DED in patientsin many respects (Dursun et al., 2002; de Paiva et al., 2006b;Niederkorn et al., 2006; de Paiva et al., 2009a). As of today, thismodel represents the best characterized animal model to study DED.

Integrins are heterodimeric glycoproteins consisting of one α- and oneβ-subunit. Expressed on the cell surface of leukocytes, integrins play arole in their recruitment to sites of inflammation. The association of aspecific α- and β-subunit determines the ligand specificity of theintegrin. The a4 integrin subunit (CD49d) is a constituent of Very LateAntigen-4, VLA-4 (integrin α4l1; CD49d/CD29) and a4137 (CD49d/CD103). Inthe case of integrin α4131, the corresponding ligands are theimmunoglobulin superfamily adhesion molecule vascular cell adhesionmolecule 1 (VCAM-1) on vascular endothelial cells and the extracellularmatrix glycoprotein fibronectin, which are responsible for the homing,trafficking, differentiation, priming, activation, proliferation andsurvival of integrin α4β1 expressing cells. Integrin α4β1 is expressedon lymphocytes, monocytes, macrophages, NK cells and eosinophils.Integrin α4β7 and its corresponding ligand, MAdCAM (Mucosal AddressinCell Adhesion Molecule-1), regulate leukocyte trafficking to the gut,but their involvement in DED cannot be ruled out.

Natalizumab, an antibody directed against the integrin α4 subunit, hasbeen shown to profoundly inhibit inflammation and improve clinicaloutcomes in both multiple sclerosis (Cross and Naismith, 2014) andCrohn's disease (Cohen et al., 2014) which are also T cell mediatedpathologies. Lifitegrast, a small molecule antagonist, directed againsta different adhesion molecule (LFA-1, integrin αLβ2), has been shown toreduce corneal staining and improve symptoms when delivered topically todry eye patients (Sheppard et al., 2014). Furthermore, a specific smallmolecule antagonist to integrin α4β1, BIO-8809, had been shown todecrease corneal fluorescein staining, conjunctival T cell infiltratesand TNFα expression in cornea and conjunctiva in a murine dry eye model(Ecoiffier et al., 2008). Taken together these considerations provided arationale for further exploring the blockade of integrin α4 in an animalmodel of DED.

SUMMARY OF THE INVENTION

In one aspect, the present application is directed to a method fortreating an ocular inflammatory condition in a mammal in need thereof,comprising ocularly administering to the mammal a therapeuticallyeffective amount of a compound of formula I

or a pharmaceutically acceptable salt, ester, anhydride, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof (collectively referred to as therapeutic agent A). Incertain embodiments, therapeutic agent A is applied topically. Topicaladministration may be to the cornea, the conjunctival sac, and/or theeyelid. In certain other embodiments, therapeutic agent A is appliedlocally, e.g., subconjunctivally, intracamerally, intravitreally,subtenon, subretinally, subchoroidally, or suprachoroidally.

In another aspect, the present application is directed to a compound offormula I

or a pharmaceutically acceptable salt, ester, anhydride, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof (collectively referred to as therapeutic agent A) foruse in treating an ocular inflammatory condition. In certainembodiments, therapeutic agent A is provided in a pharmaceuticallyacceptable vehicle, such as a pharmaceutically acceptable ophthalmicvehicle.

In another aspect, the present application is directed to apharmaceutical composition comprising a compound of formula I

or a pharmaceutically acceptable salt, ester, anhydride, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof (collectively referred to as therapeutic agent A) andone or more pharmaceutically acceptable excipients, wherein thecomposition is useful for treating an ocular inflammatory condition. Incertain embodiments, the pharmaceutical composition is suitable forocular administration. In some such embodiments, the pharmaceuticalcomposition is suitable to be applied topically. In certain embodiments,the pharmaceutical composition is suitable to be applied to theconjunctival sac or to the eyelid. In certain other embodiments, thepharmaceutical composition is suitable to be applied subconjunctivally,intracamerally, intravitreally, subtenon, subretinally, subchoroidally,or suprachoroidally.

In another aspect, the present application is directed to apharmaceutical composition comprising therapeutic agent A and one ormore pharmaceutically acceptable excipients, wherein the composition isuseful for treating an ocular inflammatory condition when applied in theform of an eye drop, spray or mist.

In a further aspect, the present application is directed to apharmaceutical composition comprising therapeutic agent A and one ormore pharmaceutically acceptable excipients, wherein the composition isuseful for treating an ocular inflammatory condition when applied as atopical ophthalmic formulation.

In still yet another aspect, the present application is directed to amethod for treating an ocular inflammatory condition in a mammal (whichmammal may be a human) in need thereof by preventing or reducing themigration of antigen-presenting cells to the lymph nodes. Such methodcomprises administering to said mammal (which mammal may be a human) atherapeutically effective amount of therapeutic agent A.

In another aspect, the present application is directed to apharmaceutical composition comprising a compound of formula I

or a pharmaceutically acceptable salt, ester, anhydride, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof and one or more pharmaceutically acceptableexcipients.

In yet another aspect, the present application is directed to a methodfor treatment of an ocular inflammatory condition, such as DED, in amammal (which mammal may be a human) in need thereof comprisingadministering to said mammal/human a therapeutically effective amountof: (a) a pharmaceutical composition comprising therapeutic agent A; (b)cyclosporin A; and (c) one or more pharmaceutically acceptableexcipients.

In still yet another aspect, the present application is directed to amethod for treatment of an ocular inflammatory condition, such as DED,in a mammal (which mammal may be a human) in need thereof comprisingadministering to said mammal/human a therapeutically effective amountof: (a) a pharmaceutical composition comprising therapeutic agent A; (b)a topical steroid selected from the group consisting of dexamethasonebase and phosphate, difluprednate, fluocinolone, fluorometholone baseand acetate, loteprednol, prednisolone acetate and phosphate,rimexolone, and triamcinolone acetonide; and (c) one or morepharmaceutically acceptable excipients.

In yet a further aspect, the present application is directed to a methodfor treatment of an ocular inflammatory condition, such as DED, in amammal (which mammal may be a human) in need thereof comprisingadministering to said mammal/human a therapeutically effective amountof: (a) a pharmaceutical composition comprising therapeutic agent A; (b)a non-steroidal anti-inflammatory drug selected from the groupconsisting of bromfenac, diclofenac, flurbiprofen, ketorolac, andnepafenac; and (c) one or more pharmaceutically acceptable excipients.

In still yet a further aspect, the present application is directed to amethod for treatment of an ocular inflammatory condition, such as DED,in a mammal (which mammal may be a human) in need thereof comprisingadministering to said mammal/human a therapeutically effective amountof: a) a pharmaceutical composition comprising therapeutic agent A; (b)an LFA-1 integrin antagonist such as lifitegrast; and (c) one or morepharmaceutically acceptable excipients.

In yet another aspect, the present application is directed to the use ofa compound of formula I

or a pharmaceutically acceptable salt, ester, anhydride, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof in the manufacture of a medicament for the treatmentof an ocular inflammatory condition.

In certain embodiments of any of the aforementioned aspects, the ocularinflammatory condition is dry eye disease. In other embodiments of eachaspect, the ocular inflammatory condition is non-infectious uveitis(anterior, intermediate, posterior, pan), non-infectious conjunctivitis,iritis, or scleritis.

In certain embodiments of any of the aforementioned aspects, therapeuticagent A is compound (I) or a pharmaceutically acceptable salt thereof.In certain embodiments, therapeutic agent A is the potassium salt ofcompound (I).

It will be understood by the skilled artisan that any of the precedingmethods of treatment and/or uses can be accomplished by the topical orother ocular administration of a pharmaceutical composition containingtherapeutic agent A. The term “topical administration” as used hereinincludes application in the form of an eye drop, spray, mist, gel, creamor ointment applied to the surface of the eye, application to theconjunctival sac, application via an insert, application via a drugdelivery device designed to deliver ophthalmic medications, and thelike. The term “other ocular administration” as used herein includessubconjunctival, intracameral, intravitreal, subtenon, subretinal,subchoroidal, or suprachoroidal application of a pharmaceuticallyacceptable formulation, insert or device designed to deliver ophthalmicmedications, and the like.

Based on the data presented herein, it is expected that blockade of α4integrin receptors will treat and ameliorate other ocular inflammatoryand immunological conditions whose pathogenesis involves leukocytes,such as all forms of uveitis (anterior, intermediate, posterior, pan),conjunctivitis, iritis, or scleritis (diffuse, nodular, necrotizing) inaddition to DED.

The compound of formula I, which is also knownas(S)-3-(4-((4-carbamoylpiperidine-1-carbonyl)oxy)phenyl)-2-((S)-4-methyl-2-(2-(o-tolyloxy)acetamido)pentanamido)propanonic acid; or(2S)-3-[4-({[4-(Aminocarbonyl)-1-piperidinyl]carbonyl}oxy)phenyl]-2-[((2S)-4-methyl-2-{[2-(2-methylphenoxy)acetyl]amino}pentanoyl)amino]propanoic acid; or GW559090, is a potent integrin α4 antagonist whichhad previously been clinically investigated in asthma patients by theoral inhalation route (Ravensberg et al., 2006).

Without being bound to any mechanistic theory of action, the effect ofthe compound of formula I appears to be both anti-inflammatory anddisease modifying. When administered locally to the eye, the compound offormula I improved the corneal epithelial barrier function, decreasedinflammatory markers in the cornea and conjunctiva, reduced therecruitment and homing of T lymphocytes to the conjunctiva, and,surprisingly, blocked the migration and activation of antigen-presentingcells to the draining lymph nodes in a murine model of DED. The compoundof formula I was ineffective when administered systemically. This issurprising in light of the well-known role of integrins in leukocytetrafficking. If blockade of leukocyte trafficking was the sole, or atleast primary, mechanism of action of an integrin α4 antagonist such asthe compound of formula i, it can be reasoned that systemic drugexposure should be more important than drug exposure at the ocularsurface. Leukocyte trafficking is inhibited by an integrin α4 antagonistbound to circulating leukocytes before they transmigrate the vascularendothelial wall at sites of inflammation through interaction with thecell adhesion molecules VCAM-1 and MAdCAM. The fact that the topicalroute of administration of the compound of formula I, but not thesystemic route, was effective in treating signs of DED in this animalmodel suggests a local rather than systemic effect by integrin o4antagonism. This local effect appears to be specific to the integrin α4antagonist the compound of formula I and differentiated from topicalsteroid treatment (dexamethasone phosphate) in that topicaldexamethasone phosphate ameliorated the DED associated corneal staining,but not the migration of antigen-presenting cells to the draining lymphnodes. It can be appreciated by one of skill in the art from thoseconsiderations that the therapeutic effect of integrin α4 antagonismemploys a unique mechanism. An important and disease-modifying aspect ofthis unique mechanism is the interruption of the immune cycle, presentin DED, at the draining lymph node level, a mechanism not shared withother drugs, such as topical steroids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: shows that topical treatment with the compound of formula Idose-dependently prevents OS-induced corneal barrier disruption.

Geometric mean±95% confidence intervals of intensity score of OGD uptakeafter 5 days of desiccating stress (DS). NS=non-stressed untreated;DS5=desiccating stress for 5 days without topical treatment; all othergroups are DS5 with topical treatment; Dex=Dexamethasone phosphate 0.1%;BSS=Balanced Salt Solution (Leiter's Pharmacy, San Jose, Calif.),vehicle for dexamethasone; V GW=phosphate-buffered saline, pH 7 (vehiclefor GW559090); GW_1 mg/mL=GW559090 (1 mg/mL); GW_3 mg/mL=GW559090 (3mg/mL); GW_10 mg/mL=GW559090 (10 mg/mL); GN_30 mg/mL=GW559090 (30mg/mL). N=26-30. *p.0.05; **p<0.01 compared to control (DS5 vs NS; Dexvs Dex vehicle; GW vs GW vehicle; mixed effects ANOVA of Log 10 OGDdata).

FIG. 2: shows that the compound of formula i acts primarily locally, notsystemically, to improve DS-induced corneal barrier disruption.

Geometric mean±95% confidence intervals of intensity score of OGD uptakeafter 5 days of desiccating stress (DS). NS=non-stressed untreated;DS5=desiccating stress for 5 days without treatment; all other groupsare DS5 with treatment; Dex=Dexamethasone phosphate 0.1%; V Dex=BalancedSalt Solution (Leiter's Pharmacy, San Jose, Calif.), vehicle fordexamethasone; GW topical=topical GW559090 (30 mg/mL; 60 μg per eye); GWSC=subcutaneous GW559090 (30 mg/mL; 120 μg); V GW=phosphate-bufferedsaline, pH 7 (vehicle for GW559090). N=26-30.

*p<0.05; **p<0.01 compared to control (DS5 vs NS; GVVW vs GW vehicle;mixed effects ANOVA of Log 10 OGD data).

FIG. 3A and FIG. 3B: show that topical treatment with the compound offormula I decreases inflammatory markers.

FIG. 3A. Relative fold of expression±SD of IL-1a, MMP-9, CXCL-9, TGF-b1genes in cornea

FIG. 3B. Relative fold of expression±SD of TGF-b1 gene in conjunctivaLine indicates non-stressed, untreated control.

V GW=phosphate-buffered saline, pH 7 (vehicle); GW_30 mg/mL=GW559090 (30mg/mL). N=7-8.

*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Unpaired T test)

FIG. 4: shows that topical treatment with the compound of formula Idecreases CD11c+ and CD11c+/MHC II+ dendritic cells in draining cervicallymph nodes.

Percent gated cells (FACS) isolated from draining CLN which were pooledfrom each mouse were stained for CD11c, CD11b and MHC II.NS=non-stressed untreated; DS1=desiccating stress for 1 day withouttreatment; all other groups are DS1 with treatment; Dex=Dexamethasonephosphate 0.1%; V GW=phosphate-buffered saline, pH 7 (vehicle fordexamethasone and GW559090); GWN topical=topical bilateral GW559090 (30mg/mL; 60 μg pereye); GWSC=GW559090 SC BID (30 mg/mL; 120 μg). N=16.*p<0.05; compared to control (DS1 vs NS; GW vs GW vehicle; 2-way ANOVAwith fixed treatment and random experiment effects followed by Dunnett'smultiple comparison procedure).

FIG. 5: shows that topical treatment with the compound of formula idecreases conjunctival T cell infiltration.

Cell density group means and 95% confidence intervals after 5 days ofdesiccating stress (DS). NS=non-stressed untreated; DS5=desiccatingstress for 5 days without treatment; all other groups are DS5 withtreatment; GW topical=topical GW559090 (30 mg/mL; 60 μg per eye); VGW=phosphate-buffered saline, pH 7 (vehicle for GW559090). N=5.

*p<0.05; ***p<0.001 compared to DS5 control (Kruskal-Wallis procedure,followed by Dunn's multiple comparisons procedure).

FIG. 6: shows conjunctival and corneal tissue concentrations of GW559090following administration of 40 μL (50 mg/mL) GW559090A. The dashed lineat approximately 4000 nM (˜2400 ng/mL) indicates the approximate tissuelevel required to achieve the IC90 concentration of ˜80 nM (˜48 ng/mL)if protein binding is 98%. The approximate tissue level required toachieve the IC50 concentration of ˜8 nM (˜5 ng/mL) if protein binding is98% is 400 nM (˜240 ng/mL). After administration of 40 μL (50 mg/mL)GW559090A, the conjunctival and corneal tissue concentrations are inexcess of 3000 ng/mL at 30 minutes and levels persisted above 1000 ng/mLin corneal tissue at 3 hours.

DETAILED DESCRIPTION OF THE INVENTION

I. Local Action of α4 Integrin Antagonist in Murine Model of DED

DED is a multifactorial ocular surface disease that is associated withsymptoms of discomfort, visual disturbance, tear film instability andinflammation of the ocular surface leading to potential damage to theocular surface tissues (Research in Dry Eye DEWS Report 2007). Itencompasses a diverse spectrum of etiologies, such as environmental,drug-induced, contact lens wear, aging. DED can also represent an ocularmanifestation secondary to a systemic autoimmune condition, such as, butnot limited to, Sjögren's syndrome, rheumatoid arthritis, systemic lupuserythematosus, graft versus host disease, Stevens-Johnson syndrome,ocular cictricial pemphigoid, sarcoidosis (reviewed by Stern et al.,2010; Zoukhri, 2006).

As an inflammatory disease of the ocular surface DED is mediated byautoreactive T cells and is associated with corneal barrier dysfunction,increased expression and levels of inflammatory cytokines andchemokines, tear film instability and discomfort. The inventor foundthat an integrin α4 antagonist, when administered locally to the eye,rescued the corneal epithelial barrier function, decreased inflammatorymarker expression in the cornea and conjunctiva, reduced conjunctival Tcell infiltration and inhibited the migration and activation ofantigen-presenting cells to the draining lymph nodes in a murine modelof DED.

Primed and activated T lymphocytes traffic from the bloodstream to sitesof inflammation with the help of adhesion receptors expressed at theircell surface that interact with corresponding adhesion molecules on thevascular endothelium. Lymphocytic integrins α4β1 (VLA-4), α4β7 and αLβ2(LFA-1) bind to endothelial VCAM-1, MAdCAM and ICAM-1, respectively. Atsites of inflammation, lymphocytic integrin receptors can interact withcertain tissue components, fibronectin in the case of α4β1, whichfurther aids in lymphocyte homing, activation and proliferation (Nojimaet al., 1990; Cox et al., 2010). The compound of formula I hashigh-affinity for α4β1 (Table 1). In cell adhesion assays it potentlyblocked cell adhesion of α4β1 to VCAM-1 and fibronectin (CS-1 domain),as well as α4β7 to MAdCAM (Table 1). The latter interaction is ofrelevance in the gut environment but has not been studied in the eye. InSjögren's patients, who have xerostomia and DED, integrin α4β1 has beendetected in T lymphocytic infiltrates in labial tissue and VCAM-1 onvascular and dendritic cells (Edwards et al., 1993).

Increased uptake of fluorescent dyes by the corneal epithelium is ahallmark of DED. It has been previously reported that corneal stainingintensity with Oregon-Green Dextran (OGD) in mice positively correlateswith a reduction in corneal barrier function after experimental DS (dePaiva et al., 2009a; de Paiva et al., 2006b). This mimics what isobserved clinically in dry eye patients in whom it is demonstrated byfluorescein staining of the cornea. Low staining scores are indicativeof dye exclusion, i.e. corneal barrier integrity. In contrast, highstaining scores are reflective of barrier dysfunction. Cornealfluorescein staining scores have been used as important endpoints fordiagnosis of DED and as an efficacy parameter in clinical trials.

Over 5 days in a controlled DS environment (as described below underMaterials and Methods) mice developed corneal barrier dysfunction,evident as punctate corneal OGD staining (as described below underMaterials and Methods), as had been reported previously (de Paiva etal., 2009a; de Paiva et al., 2006b). In this murine model, topicaladministration of the compound of formula I dose-dependently reducedcorneal OGD staining similar to a topical corticosteroid, dexamethasonephosphate (FIG. 1). Topical steroids are effective medications for DEDif lubricants and non-steroidal immunomodulators are not effective, butthey are typically used short-term because of the potential to developsteroid-related ocular adverse events. A similar improvement of cornealstaining in this animal model with a different integrin α4β1 antagonist,BIO-8809, had been previously reported (Ecoiffier et al., 2008).Relative to body weight, topical application of a drug to the mouse eyefar exceeds the dose given to a human by 2-3 orders of magnitude. It isconceivable that topical treatment in the mouse achieves relevantsystemic exposure. Since integrins play a role in leukocyte traffickingit can be reasoned that systemic exposure may be the prerequisite for anintegrin antagonist to treat ocular disease. Thus, it was important todetermine whether systemic treatment was more effective. Interestingly,when comparing systemic with topical treatment of the same doseside-by-side the compound of formula I improved corneal stainingsignificantly only when administered topically (FIG. 2). This surprisingfinding suggests that there is a critically important local component totherapy in this disease using the compound of formula I which isrequired for therapeutic success.

It has been suggested that DED is the consequence of an immune cyclethat involves the migration of antigen-presenting dendritic cells (oftenreferred to as APC or DC) from the ocular surface to the drainingcervical lymph nodes (CLN) where the priming of autoreactive T cellstakes place (Pflugfelder et al., 2008). These autoreactive CD4+ T cellsthen home back to the ocular surface propagating the disease (Coursey etal., 2013; Niederkorn et al., 2006; Zhang et al., 2012). With integrinreceptors present on CD4+ T cells the question arose whether treatmentwith an integrin α4 antagonist affected T cells in the draining lymphnodes. One day of DS neither increased CD4+ or CD8+ T cells in draininglymph nodes, nor did treatment with the compound of formula I ordexamethasone decrease the number of T cells. As previously reported, 1day of DS significantly elevated CD11b+ monocytes in draining lymphnodes (Schaumburg et al., 2011; Zhang et al., 2014). But neither of thedrug treatments was able to reduce the number of monocytes.

It has been previously shown that DC are important for the immunemediated pathology induced by DS, as DC-depleted mice do not develop DED(Schaumburg et al., 2011). CD11c+DC appeared elevated in draining lymphnodes, albeit not significantly, by short-term DS. In contrast totopical dexamethasone or systemic treatment with the compound of formulai, which had no significant effect on these cells, activated (MHC-II+)and non-activated CD11c+ cells were decreased by topical treatment withthe compound of formula i (FIG. 4). This surprising finding suggeststhat the compound of formula I prevents the migration ofantigen-presenting cells to the draining lymph nodes and that thiseffect requires drug present at the ocular surface. Similarly, asdiscussed earlier only topical treatment with the compound of formula Iimproved corneal staining. Taken together, but without being bound byany particular theory, these results implicate that the compound offormula I acts locally at the level of the ocular surface to treatocular inflammation related to DED by preventing the migration ofantigen-presenting DC to the draining CLN, thus interrupting the immunecycle.

The pro-inflammatory milieu at the ocular surface in DED and this murinemodel is well described in the literature (Corrales et al., 2006;Coursey et al., 2014; de Paiva et al., 2009a; de Paiva et al., 2006a; dePaiva et al., 2009b; de Paiva et al., 2011). The expression of manycytokines and chemokines is increased in the cornea and conjunctivaresulting in elevated levels in the tear film. In the Examples presentedherein, topical treatment with the compound of formula I inhibited theexpression of IL-1α, MMP-9, CXCL-9 and TGFβ1 in the corneal epithelium,and of TGFβ1 in the conjunctiva (FIG. 3). IL-1α is a pro-inflammatorycytokine that is released by epithelium and inflammatory cells. Itspotential relevance for the disease is highlighted by the clinicaldevelopment of an IL-1 receptor antagonist for ocular surfaceinflammation, EBI-005 (Hou et al., 2013; Goldstein et al., 2015). MMP-9is a protease that has been implicated in the breakage of tightjunctions of corneal epithelium and in the corneal barrier disruption inDS (de Paiva et al., 2006b; Luo et al., 2004; Pflugfelder et al., 2005).Tear levels of MMP-9 have been shown to correlate with corneal stainingintensity and other clinical parameters in dry eye patients(Chotikavanich et al., 2009). CXCL9, together with CXCL10 and CXCL11,attract interferon-gamma producing Th1 cells and are elevated in thetear film and conjunctiva in dry eye patients (Yoon et al., 2010).TGF-β1 is involved in Th-17 priming together with IL-6 and IL-23 and itis found elevated in tears of dry eye patients (Gutcher et al., 2011;Stockinger et al., 2007; Zheng et al., 2010). Thus, treatment with thecompound of formula I reduces some inflammatory markers in this animalmodel that are associated with ocular surface inflammation in DED.

The Examples demonstrate an improvement in objective signs of dry eye byuse of the compound of formula I in the murine DS model. The potentintegrin α4 antagonist acted locally at the level of the ocular surfacepreventing the migration of antigen-presenting cells to the draininglymph nodes with a resulting interruption of the immune cycle of dryeye.

Treatment of this disease by blockade of antigen-presenting cellmigration represents a novel and previously unknown mechanism of actionfor integrin antagonists.

II. Methods of Treatment

The term “treatment” or “treating”, with respect to treatment of ocularinflammatory conditions, including DED, refers to, inter alia,preventing the development of the disease, or altering the course of thedisease (for example, but not limited to, slowing the progression of thedisease), or reversing a symptom of the disease or reducing one or moresymptoms and/or one or more biochemical markers in a subject, preventingone or more symptoms from worsening or progressing, promoting recoveryor improving prognosis, and/or preventing disease in a subject who isfree there from as well as slowing or reducing progression of existingdisease. For a given subject, improvement in a symptom, its worsening,regression, or progression can be determined by an objective orsubjective measure. Prophylactic methods (e.g., preventing or reducingthe incidence of relapse) are also considered treatment.

In any subject, an assessment may be made as to whether the subject has,or is at risk of having, an ocular inflammatory condition. For example,fluorescein staining of the cornea is used to diagnose dry eye disease.The assessment may indicate an appropriate course of therapy, such aspreventative therapy, maintenance therapy, or modulative therapy.

Accordingly, provided herein is a method of treating, preventing,modulating, or attenuating an ocular inflammatory condition byadministering to the subject a therapeutically effective amount of atherapeutic agent. The therapeutic agent is a compound of formula I or apharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof. Incertain embodiments, the subject is a mammal, such as a human or othermammal.

In certain embodiments, the administration is ocular, such as topicaladministration or other ocular administration (e.g., local injection).In certain embodiments, the therapeutic agent is delivered to the ocularsurface. In certain embodiments, the therapeutic agent is administeredtopically, e.g., to the cornea, conjunctiva, and/or the eyelid. Incertain embodiments, the therapeutic agent is administered topically tothe cornea. In certain embodiments, the therapeutic agent is applied tothe conjunctival sac or to the eyelid. In certain embodiments, thetopical administration involves application of eye drops, ointments, orlotions. In certain embodiments, the therapeutic agent is appliedsubconjunctivally, intracamerally, intravitreally, subtenon,subretinally, subchoroidally, or suprachoroidally. In certainembodiments, the therapeutic agent is delivered via local injection,such as, for example, periocular, intraocular, subconjunctival,retrobulbar, or intracameral injection. While systemic administration isnot preferred, in certain embodiments, the administration may besystemic.

In certain embodiments, administration is achieved by insertion of asustained release device, such as a mini- or micropump, that releases atherapeutic agent. The sustained release device may be bio-degradable ornon-bio-degradable.

A therapeutic agent may be delivered in a pharmaceutically acceptableophthalmic vehicle, such that the therapeutic agent is maintained incontact with the ocular surface for a sufficient time period to allowthe therapeutic agent to penetrate the corneal and internal regions ofthe eye. The pharmaceutically acceptable ophthalmic vehicle may, forexample, be an ointment, vegetable oil or an encapsulating material.

In certain embodiments, the therapeutic agent acts locally to interruptan immune cycle involving migration of antigen-presenting cells from anocular surface to a draining lymph node. In certain particularembodiments, the therapeutic agent acts locally to block migration ofantigen-presenting cells to a draining lymph node. In certainembodiments, the draining lymph node is a cervical lymph node.

In certain embodiments, the ocular inflammatory condition is dry eyedisease, non-infectious uveitis (including anterior, intermediate,posterior, and pan uveitis), non-infectious conjunctivitis, iritis, orscleritis

In certain embodiments where the ocular inflammatory condition is dryeye disease, the dry eye disease is caused by, or associated with,allergies, diabetes, lacrimal gland deficiency, systemic lupuserythematosus, graft versus host disease, Parkinson's disease, Sjögren'ssyndrome, rheumatoid arthritis, complications arising from LASIK therapyfor vision correction, contact lens use, exposure to arid climates, airpollution, or cigarette smoke, corneal injury, conjunctival fibrosis,Stevens-Johnson syndrome, congenital alachrima, ocular cictricialpemphigoid, sarcoidosis, or treatment with other drugs that causesymptoms of dry eye disease. In certain embodiments, the ocularinflammatory condition is dry eye disease associated with Sjögren'ssyndrome. Thus, in one embodiment, the methods comprise administeringthe therapeutic agent to a human having allergies, diabetes, lacrimalgland deficiency, systemic lupus erythematosus, graft versus hostdisease, Parkinson's disease, Sjdgren's syndrome, rheumatoid arthritis,complications arising from LASIK therapy for vision correction, contactlens use, exposure to arid climates, air pollution, or cigarette smoke,corneal injury, conjunctival fibrosis, Stevens-Johnson syndrome,congenital alachrima, ocular cictricial pemphigoid, sarcoidosis, or whohas been treated with other drugs that cause symptoms of dry eyedisease.

In general, the dosage of therapeutic agent will vary depending uponsuch factors as the subject's age, weight, height, gender, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with an individual dose of thetherapeutic agent in the range of from about 0.001 mg to about 3000 mg,more particularly about 0.01 mg to about 300 mg, more particularly about0.1 mg to about 30 mg, more particularly about 0.5 mg to about 10, moreparticularly about 1 mg to about 5 mg, in each affected eye. In certainembodiments, an individual dose of the therapeutic agent is about 0.6,about 0.8, about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2,about 3.4, about 3.6, about 3.8, or about 4.0 mg in each affected eye.In certain embodiments, an individual dose of the therapeutic agent isabout 1.8 mg in each affected eye. In certain embodiments, an individualdose of the therapeutic agent is about 3.0 mg in each affected eye. Itis to be noted that dosage values may vary with the type and severity ofthe condition to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

For purposes of treatment, including prophylaxis, the therapeutic agentis administered to a subject in a therapeutically effective amount in apharmaceutically acceptable carrier. A “therapeutically effectiveamount” is one that is physiologically significant. The therapeuticagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient subject.

In certain embodiments, the pharmaceutical composition contains fromabout 1 mg/mL to about 100 mg/mL of the therapeutic agent. In certainembodiments, the pharmaceutical composition contains from about 10 mg/mLto about 100 mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains from about 10 mg/mL to about 50mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains about 10 mg/mL of the therapeuticagent. In certain embodiments, the pharmaceutical composition containsabout 15 mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains about 20 mg/mL of the therapeuticagent. In certain embodiments, the pharmaceutical composition containsabout 25 mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains about 30 mg/mL of the therapeuticagent. In certain embodiments, the pharmaceutical composition containsabout 35 mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains about 40 mg/mL of the therapeuticagent. In certain embodiments, the pharmaceutical composition containsabout 45 mg/mL of the therapeutic agent. In certain embodiments, thepharmaceutical composition contains about 50 mg/mL of the therapeuticagent.

In certain embodiments, the pharmaceutical composition is administeredin a volume from about 10 μL to about 100 μL. In certain embodiments,the pharmaceutical composition is administered in a volume from about 20μL to about 80 μL. In certain embodiments, the pharmaceuticalcomposition is administered in a volume of about 20 μL. In certainembodiments, the pharmaceutical composition is administered in a volumeof about 30 μL. In certain embodiments, the pharmaceutical compositionis administered in a volume of about 40 μL. In certain embodiments, thepharmaceutical composition is administered in a volume of about 50 μL.In certain embodiments, the pharmaceutical composition is administeredin a volume of about 60 μL. In certain embodiments, the pharmaceuticalcomposition is administered in a volume of about 70 μL. In certainembodiments, the pharmaceutical composition is administered in a volumeof about 80 μL.

In certain embodiments, the pharmaceutical composition is administeredto each affected eye at least once per day. In certain embodiments, thepharmaceutical composition is administered to each affected eye at leasttwice per day. In certain embodiments, the pharmaceutical composition isadministered to each affected eye at least three times per day. Incertain embodiments, the pharmaceutical composition is administered toeach affected eye at least four times per day. In certain embodiments,the pharmaceutical composition is administered to each affected eye atleast five times per day. In certain embodiments, the pharmaceuticalcomposition is administered to each affected eye at least six times perday. In certain embodiments, the pharmaceutical composition isadministered to each affected eye at least seven times per day. Incertain embodiments, the pharmaceutical composition is administered toeach affected eye at least eight times per day. In certain embodiments,the pharmaceutical composition is administered to each affected eye atleast nine times per day. In certain embodiments, the pharmaceuticalcomposition is administered to each affected eye at least ten times perday.

In certain embodiments, the therapeutically effective amount issufficient to achieve a tissue concentration of at least about 240 ng/mLfor at least one, at least two, at least three, at least four, at leastfive, or at least six hour(s) following administration. In certainembodiments, the tissue is a conjunctiva. In certain embodiments, thetissue is cornea. Thus, in certain embodiments, the therapeuticallyeffective amount is sufficient to achieve a conjunctival or cornealtissue concentration of at least about 240 ng/mL for at least one, atleast two, at least three, at least four, at least five, or at least sixhour(s) following administration. In certain embodiments, thetherapeutically effective amount is sufficient to achieve conjunctivalor corneal tissue concentrations of at least about 240 ng/mL for atleast six hours following administration.

In certain embodiments, the therapeutically effective amount issufficient to achieve a tissue concentration of at least about 358 ng/mLfor at least one, at least two, at least three, at least four, at leastfive, or at least six hour(s) following administration. In certainembodiments, the tissue is a conjunctiva. In certain embodiments, thetissue is cornea. Thus, in certain embodiments, the therapeuticallyeffective amount is sufficient to achieve a conjunctival or cornealtissue concentration of at least about 358 ng/mL for at least one, atleast two, at least three, at least four, at least five, or at least sixhour(s) following administration. In certain embodiments, thetherapeutically effective amount is sufficient to achieve conjunctivalor corneal tissue concentrations of at least about 358 ng/mL for atleast six hours following administration.

In certain embodiments, the therapeutically effective amount issufficient to achieve a tissue concentration of at least about 1000ng/mL for at least one, at least two, at least three, at least four, atleast five, or at least six hour(s) following administration. In certainembodiments, the tissue is a conjunctiva. In certain embodiments, thetissue is cornea. Thus, in certain embodiments, the therapeuticallyeffective amount is sufficient to achieve a conjunctival or cornealtissue concentration of at least about 1000 ng/mL for at least one, atleast two, at least three, at least four, at least five, or at least sixhour(s) following administration. In certain embodiments, thetherapeutically effective amount is sufficient to achieve conjunctivalor corneal tissue concentrations of at least about 1000 ng/mL for atleast three hours following administration.

In certain embodiments, the therapeutically effective amountadministered ocularly results in a biologically insignificant systemicexposure to the therapeutic agent. In certain embodiments, thetherapeutically effective amount administered ocularly does not producesystemic immune suppression.

The disclosed therapeutic agents and/or pharmaceutical compositionscontaining such therapeutic agents may be administered for any suitableperiod such as at least about 12 weeks, at least about 24 weeks, atleast about 36 weeks, or at least about 48 weeks. In certainembodiments, the therapeutic agent is administered for at least 12consecutive weeks. In certain embodiments, the therapeutic agent isadministered for at least 24 consecutive weeks. In certain embodiments,the therapeutic agent is administered for at least 36 consecutive weeks.In certain embodiments, the therapeutic agent is administered for atleast 48 consecutive weeks.

In certain embodiments, the treatment comprises daily administration ofthe therapeutic agent for at least two consecutive weeks. In such atreatment regimen, the therapeutic agent may be administered more thanonce daily, such as two, three, four, five, six, seven, eight, nine,ten, eleven, or twelve times per day. In certain embodiments, theduration of the treatment regimen exceeds two weeks. In certainembodiments, the duration of the treatment regimen exceeds three weeks.In certain embodiments, the duration of the treatment regimen exceedsfour weeks. In certain embodiments, the duration of the treatmentregimen exceeds five weeks. In certain embodiments, the duration of thetreatment regimen exceeds six weeks. In certain embodiments, theduration of the treatment regimen exceeds seven weeks. In certainembodiments, the duration of the treatment regimen exceeds eight weeks.In certain embodiments, the duration of the treatment regimen exceedsnine weeks. In certain embodiments, the duration of the treatmentregimen exceeds ten weeks. In certain embodiments, the duration of thetreatment regimen exceeds eleven weeks. In certain embodiments, theduration of the treatment regimen exceeds twelve weeks.

In certain embodiments, the therapeutic agent (e.g., therapeutic agentA) is co-administered with one or more additional therapeutic agents inthe same or separate pharmaceutical compositions. Such additionaltherapeutic agents may include other therapeutic agents used to treatocular inflammatory conditions.

In certain embodiments, the additional therapeutic agent is cyclosporinA. Cyclosporin A is a cyclic peptide of eleven amino acids, synthesizedby a microscopic fungus Tolypocladium inflatum. Cyclosporin A has theformula[R—[[R*,R*-(E)]]-cyclic(L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-3-hydroxy-N,4-dimethyl-L-2-amino-6-octenoyl-L-a-amino-butyryl-N-methylglycyl-N-methyl-L-leucyl-L-valy-N-methyl-L-leucyl)(CAS number 59865-13-3).

In certain embodiments, the additional therapeutic agent is a steroid.In some such embodiments, the steroid is a glucocorticoid. In some suchembodiments, the steroid is difluprednate, prednisolone, dexamethasone,fluocinolone, fluorometholone, loteprednol, medrysone, rimexolone,triamcinolone, cortisone, or hydrocortisone. In certain embodiments, thesteroid is dexamethasone (such as dexamethasone base or dexamethasonephosphate), difluprednate, fluocinolone, fluorometholone (such asfluorometholone base or fluorometholone acetate), ioteprednol,prednisolone (such as prednisolone acetate or prednisolone phosphate),rimexolone, or triamcinolone (such as triamcinolone acetonide).

In certain embodiments, the additional therapeutic agent is anon-steroidal anti-inflammatory agent. In some such embodiments, thenon-steroidal anti-inflammatory agent is selected from the groupconsisting of bromfenac, diclofenac, flurbiprofen, ketorolac, andnepafenac.

In certain embodiments, the additional therapeutic agent is a LFAantagonist, such as lifitegrast. Lifitegrast is also known as(2S)-2-[[2-(1-benzofuran-6-carbonyl)-5,7-dichloro-3,4-dihydro-1H-isoquinoline-6-carbonyl]amino]-3-(3-methylsulfonylphenyl)propanoicacid and is identified in WO2006/125119, the entire contents of whichare herein incorporated by reference.

In certain embodiments, the therapeutic agent can be used in veterinarymedicine, particularly in the treatment of ocular inflammatoryconditions such as, for example, keratoconjunctivitis sicca in dogs,cats, and horses; chronic superficial keratitis (CSK) in dogs, cats, andhorses; and lymphoplasmocytic infiltration of the nictitating membranein dogs, cats, and horses.

III. Compounds

The compound of formula I can be synthesized using the synthesisdescribed in U.S. Pat. No. 6,867,192, Example 27. The entire contents ofU.S. Pat. No. 6,867,192 is hereby incorporated by reference.

Briefly, in one embodiment,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.4 g) and1-hydroxybenzotriazole (0.3 g) are added to a solution of(2-methylphenoxy)acetic acid (0.345 g) in acetonitrile (50 ml), under anitrogen atmosphere. After stirring for 30 minutes at 20° C.,4-[(2S)-2-{[(2S)-2-Amino-4-methylpentanoyl]amino}-3-(tert-butoxy)-3-oxopropyl]phenyl4-(aminocarbonyl)-1-piperidinecarboxylate hydrochloride (1 g) is addedfollowed by diisopropylethylamine (0.35 ml) and stirring is continuedfor 18 h. The mixture is concentrated in vacuo and the residuepartitioned between 1M hydrochloric acid (100 ml) and ethyl acetate (300ml). The layers are separated and the organic phase is washed with 1Mhydrochloric acid (2×100 ml), saturated aqueous sodium hydrogencarbonate (3×100 ml) and brine (100 ml), dried over magnesium sulphateand evaporated in vacuo to give a white solid. To a solution of this inchloroform (5 ml) is added trifluoroacetic acid (5 ml) and water (1 ml).After stirring for 3 h at 2000, the solvent is evaporated in vacuo andthe residue is azeotroped with toluene (2×20 ml) then triturated withether to(2S)-3-[4-({[4-(Aminocarbonyl)-1-piperidinyl]carbonyl}oxy)phenyl]-2-[((2S)-4-methyl-2-{[2-(2-methylphenoxy)acetyl]amino}pentanoyl)amino]propanoicacid.

Alternatively, in another embodiment, a solution of(2S)-3-[4-(allyloxy)phenyl]-2-[(tert-butoxycarbonyl)amino]propanoic acid(115.8 g) and 1-hydroxybenzotriazole (48.6 g) in DMF (475 ml) is addedto Wang resin (50 g). After 15 minutes 1,3-diisopropylcarbodiimide (56.5ml) is added and the mixture is stirred for 24 h at 45° C. The resin isfiltered and washed with DMF (3×360 ml), methanol (3×360 ml) anddichloromethane (3×700 ml). To a slurry of the resin in dichloromethane(644 ml) is added pyridine (14.7 ml). Acetic anhydride (26.9 ml) isadded and the mixture is stirred for 12 h at 20° C. The resin isfiltered and washed with dichloromethane (3×550 ml), methanol (3×370 ml)and dichloromethane (3×550 ml).

A slurry of 20 g of the resin in dichloromethane (100 ml) was cooled to2-5° C. and treated with a solution of phenol (20 g) in dichloromethane(80 ml). Chlorotrimethylsilane (20 ml) is added dropwise and the mixtureis stirred for 6 h at 2-5° C. The resin is filtered and washed withdichloromethane (3×200 ml), methanol (3×200 ml), 10% water in DMF (2×200ml), 10% diisopropylethylamine in DMF (3×200 ml), DMF (200 ml), methanol(3×200 ml) and dichloromethane (3×200 ml).

A slurry of the resin in DMF (55 ml) is treated with a solution ofFmoc-leucine (32.7 g) and 1-hydroxybenzotriazole (12.5 g) in DMF (85ml). After 5 minutes 1,3-diisopropylcarbodiimide (19.3 ml) is added andthe mixture is stirred for 15 h at 20° C. The resin is filtered andwashed with DMF (3×150 ml), methanol (3×150 ml) and dichloromethane(3×150 ml).

The resin is treated with 20% piperidine in DMF (180 ml) and stirred for1 h at 20° C. The resin is filtered and washed with DMF (3×150 ml),dichloromethane (3×150 ml), DMF (3×150 ml) and dichloromethane (3×150ml). To a slurry of this in DMF (50 ml) is added a solution of(2-methylphenoxy)acetic acid (17.9 g) and 1-hydroxybenzotriazole (14.6g) in DMF (100 ml).

After 5 minutes 1,3-diisopropylcarbodiimide (16.9 ml) is added and themixture is stirred for 65 h at 200° C. The resin is filtered and washedwith DMF (2×150 ml), methanol (3×150 ml) and dichloromethane (3×150 ml).

A slurry of the resin in dichloromethane (60 ml) is treated with asolution of tetrakis(triphenylphosphine)palladium(0) (5.21 g) indichloromethane (140 ml) followed by morpholine (13 ml). The mixture isstirred for 2 h at 20° C. then the resin is filtered and washed withdichloromethane (7×200 ml).

A slurry of the resin in dichloromethane (160 ml) is treated withdiisopropylethylamine (12.4 ml) followed by 4-nitrophenyl chloroformate(24.8 g) in 3 portions at 5 minute intervals. The mixture is stirred for1 h at 200° C. The resin is filtered and washed with dichloromethane(3×200 ml). The resin is treated with a solution of isonipecotamide(15.8 g) in DMF (180 ml) and the mixture is stirred for 1.5 h at 20° C.The resin is filtered and washed with DMF (4×200 ml) and dichloromethane(2×200 ml).

The resin is treated with 50% TFA in dichloromethane (200 ml). Afterstirring for 1 h at 20° C. the resin is filtered and washed withdichloromethane (5×200 ml). The combined filtrate and washings wereevaporated in vacuo. The residue is azeotroped with toluene (2×100 ml)then triturated with ether (50 ml) and the resulting white solidfiltered. To this is added acetonitrile (150 ml) and the mixture isheated to reflux. The resulting suspension is allowed to cool to 20° C.and stirred for 18 h. The mixture is filtered to give(2S)-3-[[4({[4-(Aminocarbonyl)-1-piperidinyl]carbonyl}oxo)phenyl]-2-[((2S)-4-methyl-2-{[2-(2-methylphenoxy)acetyl]amino}pentanoyl)amino]propanoicacid.

In certain embodiments, the therapeutic agent (e.g., therapeutic agentA) is a pharmaceutically acceptable salt of compound (I). The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

In certain embodiments, the therapeutic agent (e.g., therapeutic agentA) is a potassium salt of compound (I).

In certain embodiments, the compound of formula (I) is administered as aconjugate, complex, or prodrug. In certain embodiments, the compound offormula (I) is administered in the form of a prodrug. Such prodrug maybe metabolized by the subject to provide the compound of formula (I).

IV. Pharmaceutical Compositions

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, for example the carrierdoes not decrease the impact of the agent on the treatment. In otherwords, a carrier is pharmaceutically inert.

The composition can be formulated according to known methods to preparepharmaceutically useful compositions, whereby a compound is mixed with apharmaceutically acceptable carrier. Sterile phosphate-buffered salineis one example of a pharmaceutically acceptable carrier. Other suitablecarriers are well known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (1995).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, emulsions, suspensions, lotions,powders, solutions, pastes, gels, sprays, mists, aerosols or oils. Incertain embodiments, the pharmaceutical composition is a solution, suchas an ophthalmic solution. In such embodiments, the ophthalmic solutionis administered to humans or non-human mammals via the topical route, inthe form of one or more drops per day in each eye.

For treatments of the eye the compositions may be applied as a topicalointment or cream. When formulated in an ointment, the active ingredientmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredient may be formulated in a creamwith an oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.Formulations to be administered to the eye will have ophthalmicallycompatible pH and osmolality. One or more ophthalmically acceptable pHadjusting agents and/or buffering agents can be included in acomposition of the invention, including acids such as acetic, boric,citric, lactic, phosphoric and hydrochloric acids; bases such as sodiumhydroxide, sodium phosphate, sodium borate, sodium citrate, sodiumacetate, and sodium lactate; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride.

Such acids, bases, and buffers can be included in an amount required tomaintain pH of the composition in an ophthalmically acceptable range.One or more ophthalmically acceptable salts can be included in thecomposition in an amount sufficient to bring osmolality of thecomposition into an ophthalmically acceptable range. Such salts includethose having sodium, potassium or ammonium cations and chloride,citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfateor bisulfite anions.

The instant compositions may be applied topically to the eye,subconjunctivally, intracamerally, intravitreally, subtenon,subretinally, subchoroidally, suprachoroidally, in the conjunctival sacor to the eyelid using an ocular delivery device or insert. Such adevice or insert may be designed for the controlled release of one ormore therapeutic agents with multiple defined release rates andsustained dose kinetics and permeability. Controlled release may beobtained through the design of polymeric matrices incorporatingdifferent choices and properties of biodegradable/bioerodable polymers(e.g. poly(ethylene vinyl) acetate (EVA), superhydrolyzed PVA),hydroxyalkyl cellulose (HPC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), polycaprolactone, poly(glycolic) acid, poly(lactic)acid, polyanhydride, of polymer molecular weights, polymercrystallinity, copolymer ratios, processing conditions, surface finish,geometry, excipient addition and polymeric coatings that will enhancedrug diffusion, erosion, dissolution and osmosis.

Formulations for drug delivery using ocular devices or inserts maycombine one or more active agents and adjuvants appropriate for theindicated route of administration. For example, the active agents may beadmixed with any pharmaceutically acceptable excipient, lactose,sucrose, starch powder, cellulose esters of alkanoic acids, stearicacid, talc, magnesium stearate, magnesium oxide, sodium and calciumsalts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, tableted orencapsulated for conventional administration. Alternatively, thecompounds may be dissolved in polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.The compounds may also be mixed with compositions of both biodegradableand non-biodegradable polymers, and a carrier or diluent that has a timedelay property. Representative examples of biodegradable compositionscan include albumin, gelatin, starch, cellulose, dextrans,polysaccharides, poly (D,L-lactide), poly (D,L-lactide-co-glycolide),poly (glycolide), poly (hydroxybutyrate), poly (alkylcarbonate) and poly(orthoesters) and mixtures thereof. Representative examples ofnon-biodegradable polymers can include EVA copolymers, silicone rubberand poly (methylacrylate), and mixtures thereof.

Pharmaceutical compositions for ocular delivery also include in situgellable aqueous composition. Such a composition comprises a gellingagent in a concentration effective to promote gelling upon contact withthe eye or with lacrimal fluid. Suitable gelling agents include but arenot limited to thermosetting polymers. The term “in situ gellable” asused herein includes not only liquids of low viscosity that form gelsupon contact with the eye or with lacrimal fluid, but also includes moreviscous liquids such as semi-fluid and thixotropic gels that exhibitsubstantially increased viscosity or gel stiffness upon administrationto the eye. See, for example, Ludwig (2005), herein incorporated byreference for purposes of its teachings of examples of polymers for usein ocular drug delivery.

V. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes. Akit may comprise one or more compounds as described herein, e.g., inpremeasured doses. A kit may optionally comprise devices for contactingcells with the compounds and instructions for use.

Devices include syringes, implantable pumps, such as mini- andrnicropumps, and other devices for ophthalmic use.

Also provided herein are therapeutic combinations comprising therapeuticagent A and another therapeutic agent (the same ones used in combinationtherapies and combination compositions) in separate dosage forms, butassociated with one another. The term “associated with one another” asused herein means that the separate dosage forms are packaged togetheror otherwise attached to one another such that it is readily apparentthat the separate dosage forms are intended to be sold and administeredas part of the same regimen. The compound and the other agent arepreferably packaged together in a blister pack or other multi-chamberpackage, or as connected, separately sealed containers (such as foilpouches or the like) that can be separated by the user (e.g., by tearingon score lines between the two containers).

In still another embodiment, the invention provides a kit comprising inseparate vessels, a) a compound of this invention; and b) anothertherapeutic agent such as those described elsewhere in thespecification.

EXAMPLES

Materials

[3H] GW559090 was custom synthesized at Amersham to contain five 3Hatoms per molecule giving a specific activity of 76 Ci/mmol.

All animal studies were conducted using C57BL/6 mice, 6 to 8 weeks old,which were purchased from Jackson Labs (Bar Harbor, Me.).

Binding and Cell Adhesion

Jurkat J6 cells (human lymphoblast cell line) were grown as a suspensionculture in RPMI 1640 supplemented with FCS (10%) and glutamine (2 mM).RPMI8866 cells (human B lymphoid cell line) were grown as a suspensionculture in RPMI 1640 supplemented with FCS (10%) and glutamine (2 mM).RBL-2H3 cells (rat basophilic cell line) were cultured in Eagle's MEMplus Earles salts supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 1× non-essential amino acids, 1 mM Na pyruvate 10% heatinactivated foetal calf serum.

J6 Saturation Assay (Filtration Assay):

Binding of the compound of formula I was characterized in human J6 cellswhich express VLA-4. J6 cells were harvested by centrifugation for 5minutes at 500 g and resuspended in assay buffer (50 mM HEPES, pH 7.5,100 mM NaCl, 2 mM glucose, 1 mM MnCl2). Each well contained 1×10⁶ cellsand either 10 μM cold GW559090, to define the NSBs (non-specificbinding), or buffer. [3H]GW559090 (0.02-50 nM) was added in a finalvolume of 500 μL and incubated for 2 h at 37° C. Bound [3H]GW559090 wasseparated from free by rapid vacuum filtration through pre-soakedWhatman GF/B filters, followed by three washes in ice-cold buffer,scintillant was then added to filter discs and disintegrations perminute measured on a Beckman Scintillation counter. The actual amount of[3H] GW559090 added for each concentration of the saturation curve wasmeasured by counting disintegrations from a 50 μL aliquot of the labeldilution range.

Saturation Binding Assay in RBL-2H3 Cells (SPA Assay):

The assay buffer contained 50 mM HEPES, 100 mM NaCl, 1 mM MnCl2, pH 7.5(with NaOH). WGA SPA beads were used at 1 mg/well. Cells were harvestedand resuspended in assay buffer and 1 million cells were added per wellin a white bottomed plate. Cold GW559090 to give a final assayconcentration of 20 μM (to define nonspecific binding) or buffer alonewere added and then [3H] GW559090 across a concentration range acrossthe plate was added (nominal [3H]GW559090 concentration range was 0.01to 200 nM). The final assay volume was 250 μL. The plate was thenincubated at 37° C. for 2 h. Disintegrations were counted byscintillation (from the WGA SPA beads) in a Wallac Microbeta platereader.

The actual amount of [3H] GW559090 added for each concentration of thesaturation curve was measured by counting disintegrations per minutefrom a 50 μL aliquot of the label dilution range.

VCAM Cell Adhesion Assay:

Polystyrene 96-well microtitre plates were coated with IgG at aconcentration of 0.05 mg/mL in bicarbonate buffer for 2 hours at 37° C.The solution was aspirated and the plates washed twice with PBS. Theplates were then incubated overnight at 4° C. with a 1:4000 dilution ofVCAM-1 of 3% BSA in PBS. Prior to use the VCAM-1 was aspirated and theplates washed twice with PBS.

J6 or RPMI cells (as required) were labelled with the fluorescent dyeBCECF-AM (10 μM and 6×10⁶ cells/mL) for 10 min at 37° C. before theexcess was removed by centrifugation at 500 g for 5 min and the cellsresuspended at a cell concentration of 1.2×10⁷ cells/mL in HBSS. Equalvolumes HBSS containing the compound of formula I (over concentrationrange 38.1 μM to 10 μM) and cells were added to the VCAM-1 coatedplates. After a 30 minute incubation at 37° C., non or loosely adheringcells were removed by inverting the plate and blotting on tissue paper.Two washes with PBS and blotting were followed by addition of TritonX-100 (2% v/v). The plates were counted in a Wallac Viktor. Compoundsthat inhibited adhesion resulted in a lower fluorescence reading.

MAdCAM Cell Adhesion Assay:

Polystyrene 96-well microtitre plates were coated with IgG at aconcentration of 0.05 mg/mL in bicarbonate buffer for 2.5 hours at 37°C. The solution was aspirated and the plates washed twice with PBS. Theplates were then incubated overnight at 4° C. with MAdCAM at aconcentration of 204.0 ng/mL in 3% BSA in PBS. Prior to use the MAdCAMwas aspirated and the plates washed twice with PBS. J6 or RPMI cells (asrequired) were labelled with the fluorescent dye BCECF-AM (10 μM and6×10⁶ cells/mL) for 10 min at 37° C. before the excess was removed bycentrifugation at 500 g for 5 min and the cells resuspended at a cellconcentration of 1.2×10⁷ cells/mL in HBSS. Equal volumes of HBSScontaining the compound of formula I (over concentration range 38.1 μMto 10 μM) and cells were added to the MAdCAM coated plates. Adhesiontook place over a 30-minute incubation at 37° C. Non or loosely adheringcells were removed by inverting the plate and blotting on tissue paper.Two washes with PBS and blotting were followed by addition of TritonX-100 (2% v/v). The plates were counted in a Wallac Viktor.

CS-1 Cell Adhesion Assay:

Polystyrene 96-well microtitre plates were coated with CS-1 (connectingsegment 1, a cell attachment domain of fibronectin) at a concentrationof 0.01 mg/mL in bicarbonate buffer overnight at 4° C. The solution wasaspirated and the plates washed twice with PBS. The plates were thenincubated at room temperature in the presence of 3% BSA for 60 min,‘flicked’ to expel the BSA and washed twice in bicarbonate buffer. J6 orRPMI cells (as required) were labelled with the fluorescent dye BCECF-AM(10 μM and 6×10⁶ cells/mL) for 10 min at 37° C. before the excess wasremoved by centrifugation at 500 g for 5 min and the cells resuspendedat a cell concentration of 1.2×10⁷ cells/mL in HBSS. Equal volumes ofHBSS containing the compound of formula I (over concentration range 19.0μM to 5 μM) and cells were added to the CS-1 coated plates. Adhesiontook place over a 30 minute incubation at 37° C. Non or loosely adheringcells were removed by inverting the plate and blotting on tissue paper.Two washes with PBS and blotting were followed by addition of TritonX-100 (2% v/v). The plates were counted in a Wallac Viktor.

Induction of Desiccating Stress, Treatment Regimen

Female C57BL/6 mice, aged 6 to 8 weeks, were subjected to DS for 5 days(DS5) as described previously (de Paiva et al., 2006b; Niederkorn etal., 2006). Topical bilateral treatment with 1 eye drop per eye (2 μLvolume) or subcutaneous injection (4 μL volume), 2 times per day, wasinitiated on day 1 concurrently with DS and continued through day 4.Treatment with the compound of formula I or dexamethasone phosphate 0.1%was compared to correspondingly treated vehicle controls. Mice wererandomized to receive one of the test articles. Control mice were keptin a non-stressed (NS) environment maintained at 50-75% relativehumidity without exposure to airflow or scopolamine and were not treatedwith test or control article. Treatment effects were assessed on cornealstaining with Oregon Green Dextran (OGD); expression of inflammatorymarkers in ocular surface tissues by real time PCR; cell populationanalysis in draining cervical lymph nodes by FACS analysis; conjunctivalT cell infiltration by immunohistochemistry.

Corneal OGD Staining

On the morning of the 5th day, mice received one s.c. dose ofscopolamine. Two hours later corneal staining was assessed using OregonGreen Dextran (OGD-488), which is a conjugated fluorescent dye of a 70kDa molecular size (Invitrogen-Molecular Probes) as previously described(de Paiva et al., 2006b). The procedure consisted of instillation of 0.5μL of OGO on the cornea using a glass capillary pipette, 1 minute beforeeuthanasia. Mice were euthanized by inhalation of isoflurane gasfollowed by cervical dislocation. Eyes were then rinsed with 2 mL ofBSS. Excess liquid was carefully blotted from the ocular surface withfilter papers without touching the cornea. Digital pictures of both eyeswere taken under 470 nm excitation and 488 nm emission wave lengthsusing a Nikon SMZ-1500 stereo microscope, with an exposure time of 2seconds. Both eyes from each animal were evaluated; the right eye alwaysfirst followed by the left eye. The mean intensity in the central corneawas evaluated from digital images using NIS Elements (version 3.0) byplacing a fixed region of interest (a 2-mm diameter circle) on thecentral cornea. The mean intensity of the fluorescence was read by thesoftware and stored in a database (Excel, Microsoft). This fluorescencemeasurement in the central ring was done independently by 2 maskedobservers, for each mouse eye. By the conclusion of the experiment,results were averaged from both observers using all data collectedduring all study weeks (Excel, Microsoft). Results are presented asgeometric mean±95% confidence interval of gray levels.

RNA Isolation and Reverse Transcription

Total RNA was isolated from corneal and conjunctival epithelia that wascollected and pooled from 2 eyes (right and left) at each time pointfrom untreated control mice, mice subjected to DS for 5 days (DS5), DS5mice topically treated with the compound of formula I (30 m/mL) andvehicle treated animals (n=7/group) using a PicoPure™ RNA Isolation Kit(Acturus Bioscience inc, Mountain View, Calif., USA) following themanufacturer's protocol. Briefly, corneal epithelial cells were scrapedand whole conjunctiva was cut and placed in 100 μL of extraction bufferand incubated at 42° C. for 30 minutes. The cell extract was loaded ontoa preconditioned purified column, which was centrifuged, treated withDNase (Qiagen, Valencia, Calif., USA) and washed twice with twodifferent wash buffers. The RNA was eluded in 12 μL of low ionicstrength buffer. The RNA concentration was measured by absorption at 260nm using a spectrophotometer (NanoDrop 2000, Thermo Scientific,Wilmington, Del., USA) and samples were stored at −80° C. until use.First-strand cDNA was synthesized from 1 μg of total RNA with randomhexamers using M-MuLV reverse transcriptase (Ready-To-Go You-PrimeFirst-Strand Beads; Amersham Pharmacia Biotech, Inc., Piscataway, N.J.)as previously described (de Paiva et al., 2006a).

Absolute Real Time Polymerase Chain Reaction

A cDNA aliquot (1-4 μL) from samples was used for real time PCR in atotal volume of 10 μL containing the following per reaction: 0.3 μL ofgene specific Taqman probes used and 5 μL of 2× Taqman Fast PCR MasterMix, (Applied Biosystems). Real time PCR was performed on a StepOnePus™Real-Time PCR System (Applied Biosystems) and the parameters consistedof pre-denaturation at 95° C. for 22 sec, followed by 40 cycles ofdenaturation at 95° C. for 1 sec, annealing and extension at 60° C. for20 sec. Samples and standards were assayed in duplicate. A non-templatecontrol and total RNA without retrotranscription were included in allthe experiments to evaluate PCR and DNA contamination of the reagentsused. The following mouse Taqman probes were used: IL-1α (Mm00439620);MMP-9 (Mm00442991); CXCL9 (Mm004434946); TGF-β1 (Mm00441724) and HPRT-1(Mm00446968). The HPRT-1 gene was used as an endogenous reference foreach reaction. The results of quantitative PCR were analyzed by thecomparative C_(T) method where target change=2-ΔΔC_(T). The results werenormalized by the C_(T) value of HPRT-1 of the untreated control group(de Paiva et al., 2006b).

Dissection of Draining Lymph Nodes

Cervical draining lymph nodes (CLN) were surgically excised, placed in around culture plate with ˜8 mL of complete RPMI media and smashedbetween two frosted slides.

Flow Cytometry

Single-cell suspensions of CLN from C57BL/6 mice, treated under DSconditions for 1 day, were prepared, centrifuged and sequentiallyfiltered, as previously described (de Paiva et al., 2009a). Subsequently1.0 mL of ACT solution was added for 30 seconds followed by 2.0 mL ofcomplete RPMI. The cells were once again centrifuged and the supernatantaspirated. Cells were then re-suspended in 2.0 mL of complete RPMI andprepared for counting. Ten μL of Trypan Blue were used for counting andthe cells were divided in tubes at 1×10⁵ cells/mL. Subsequently, thecells were incubated on ice in 20 μL unconjugated anti-mouse CD16/CD32(BD Pharmingen, San Diego, Calif.) followed by 80 μL of directlyconjugated primary antibody or isotype. Finally the samples were washedwith 1 mL PBS/1% FBS, centrifuged and re-suspend in 0.3 mL PBS/1% FBScontaining 1:1000 of PI. The samples were stored at 4° C. until theanalysis was performed with an A BD LSRII Benchtop cytometer. The datawas analysed using BD Diva Software (BD Pharmingen) and FlowJo (TreeStarInc).

Conjunctival T Cell Infiltration

For immunohistochemistry, the eyes and adnexa of 5 mice/group (n=5) wereexcised, embedded in optimal cutting temperature (OCT compound; VWR,Suwanee, Ga.), and flash frozen in liquid nitrogen. Sagittal 8-μmsections were cut with a cryostat (HM 500; Micron, Waldorf, Germany) andplaced on glass slides that were stored at −80° C. Immunohistochemistrywas performed to detect and count the cells in the conjunctivalepithelium and stroma that stained positively for CD4 (clone H129.9, 10ug/mL, BD Bioscience, San Diego, Calif.). Cryosections were stained withthe above mentioned primary antibody and appropriate biotinylatedsecondary antibody (BD Pharmingen) and Vectastain Elite ABC usingNovaRed reagents (Vector, Burlingame, Calif.). Secondary antibody aloneand appropriate anti-mouse isotype (BD Biosciences) were also used ascontrols. Two sections from each animal were examined and photographedwith a microscope equipped with a digital camera (Eclipse E400 with aDS-Fil; Nikon). Positively stained cells were counted in the goblet cellrich area of the conjunctiva, over a length of at least 500 μm in theepithelium for a distance of 500 μm using image-analysis software (NISElements Software, version 3.0, BR, Nikon).

Statistical Analysis

Due to skewed distribution of OGD data, the analysis was done on the log10 scale to make the data more normally distributed. Log 10 OGD data wasaveraged across two observers and the left and right eyes. The mixedeffects ANOVA model included treatment group as a fixed effect and weekas a random effect. 95% and 99% confidence intervals were constructedfor the disease (5 day DS vs untreated group kept in separate vivarium)and compound treatment effects (treatment vs vehicle). As thesecomparisons were all pre-planned (comparing each treatment to itsvehicle group), no adjustment was made for multiplicity. Log 10 scaletreatment effect estimates and confidence limits were converted back tothe original OGD scale, thus representing the estimate for the ratio ofgroup geometric means and their confidence limits. For gene expressionanalysis, an unpaired T test was performed to compare drug and vehicletreatment. For flow cytometry analysis, a 2-way ANOVA with fixedtreatment and random experiment effects was employed to test thetreatment differences. The analysis was followed by Dunnett's multiplecomparison procedure to compare each of the treatment groups with thevehicle group. T cell density was calculated for each section andaveraged across sections for each animal. As the distribution of averagecell density data was non-normal, it was analyzed by usingnon-parametric methods (Kruskal-Wallis procedure, followed by Dunn'smultiple comparisons technique).

Example 1. Pharmacology of GW559090

[3H]GW559090 binding to human J6 cells was saturable and was describedin these experiments by a single binding site with mean Kd of 0.19 nM(0.08-0.43) (geometric mean and 95% CL) derived from 4 separateexperiments. A single high affinity binding site for [3H]GW559090 wasalso shown in rat RBL-2H3 cells which express rat α4β1, mean Kd 1.04 nM(0.58-1.89).

Inhibition of cell adhesion was determined for α4β1 (Jurkat J6 cells) toVCAM-1 and CS-1 (fibronectin domain); for α4β7 (RPMI 8866 cells) toMAdCAM in coated microtiter plates. GW559090 inhibited J6 cell adhesionto VCAM-1 in a monophasic fashion with a mean IC50 of 7.72 nM(2.39-24.9). GW559090 also inhibited J6 cell adhesion to CS-1 with amean IC50 of 8.04 nM (3.05-21.2) and to MAdCAM in a biphasic manner,supporting the presence of a high and low affinity site forMAdCAM—GW559090 binding in J6 cells. The RPMI 8866 MAdCAM bindingpredominantly measures α4β7 mediated cell adhesion. GW559090 inhibitedRPMI 8866 cell adhesion to MAdCAM with an IC50 of 23.0 nM (200-26.4).GW559090 also inhibited RPMI 8866 binding to VCAM-1, and CS-1 in asimple monophasic manner with respective IC50s of 4.81 nM (2.82-8.20)and 24.5 nM (identical duplicate values).

TABLE 1 Binding of [3H] GW559090 to α4 integrins and inhibition of celladhesion Binding (Kd) Human α4β1 0.19 nM Rat α4β1 1.04 nM Inhibition ofcell α4β1 - VCAM-1 7.72 nM adhesion (IC50) α4β1 - CS-1 8.04 nM α4β7 -MAdCAM 23.0 nM

Additionally, the compound of formula I had been reported as highlyselective vs. non-4 integrins, including LFA-1 (Ravensberg et al.,2006). No significant inhibition was observed by the compound of formulaI (at 10 μM) in radioligand binding assays on 53 receptors and 4transporters in an MDS Pharma screen.

Example 2. Topical Treatment with the Compound of Formula I PreventsDesiccation-Induced Corneal Barrier Disruption

We found a significant increase in corneal permeability measured by OGDstaining between the untreated non-stressed (NS) and dry eye controlgroups (DS5, FIG. 1). Because corticosteroid therapy has been reportedto improve corneal epithelial disease of dry eye both in humans and mice(de Paiva et al., 2006a; de Paiva et al., 2006b; Marsh and Pflugfelder,1999), we used topical treatment with Dexamethasone (Dex) as positivecontrol. Treatment with 0.1% Dex significantly improved OGD intensityscores. A range of doses of the compound of formula I, as low as 1 mg/mLand as high as 30 mg/mL, was investigated. A significant decrease in OGDintensity scores was noted with increasing concentrations of thecompound of formula I vs. its vehicle. The most efficaciousconcentration was 30 mg/mL (FIG. 1) which was therefore used insubsequent studies.

Example 3. The Compound of Formula I Acts Topically on the OcularSurface

To explore the effect of systemic administration of the compound offormula I on corneal staining, two routes of administration, topical andsubcutaneous (SC), were compared. An identical dose was given SC (120 μgas one 4 μL bolus) and topically (60 μg as a 2 μL drop to each eye).Mice receiving the compound of formula I systemically were also givenvehicle eye drops topically. As shown in FIG. 2 and similar to FIG. 1,topical dexamethasone decreased OGD uptake. Topical treatment with thecompound of formula I again significantly decreased DS-induced cornealbarrier disruption compared to its vehicle. This effect was not observedwhen the compound of formula I was administered systemically. Theseresults indicate that the local therapeutic effect of the compound offormula I is achieved by topical administration as traditional eyedrops. This represents a surprising finding given the known role ofintegrins in leukocyte trafficking from the bloodstream which shouldrequire systemic drug exposure. This local action from topicallyadministered drug is suggestive of a novel mechanism of action for an α4integrin antagonist.

Example 4. Topical Treatment with the Compound of Formula I DecreasesInflammatory Markers on the Ocular Surface

DED is often accompanied by increased T-cell related cytokines,matrix-metalloproteinases and inflammatory cytokines in cornea andconjunctiva (Coursey et al., 2014; de Paiva et al., 2009a; Yoon et al.,2010). We investigated the expression of IL-1α, TGF-β1, MMP-9 and CXCL-9in cornea and conjunctiva using mice that were topically treated withthe compound of formula I at 30 mg/mL during 5 days of DS and comparedto vehicle dosed mice. These genes were chosen since they are highlyinducible by DS (Coursey et al., 2014; de Paiva et al., 2009a; de Paivaet al., 2006b; de Paiva et al., 2006a; Yoon et al., 2007). Bothexperimental groups and non-stressed controls had similar expression ofthe housekeeping gene; experimental groups were calibrated tonon-stressed control. The results are presented in FIG. 3.

There was a significant decrease in IL-1α, MMP-9, TGF-β1 and CXCL9transcripts in corneas treated with GW559090 compared to vehicle control(FIG. 3A). In conjunctiva, there was a significant reduction in TGF-β1,expression but no change regarding IL-1α, MMP-9 and CXCL-9 (FIG. 3B).These results indicate that the compound of formula I can decreasemarkers of inflammation at the ocular surface.

Example 5. Topical Treatment with the Compound of Formula I DecreasesDendritic Cell Activation

The draining CLN are an integral part of the immune cycle in DED(Pflugfelder et al., 2008; Schaumburg et al., 2011). To determine theimpact of the compound of formula i, if any, on the CLN, mice weresubjected to OS for 1 day and treated either topically or systemically(subcutaneous; identical 120 μg dose as topical) twice-daily with thecompound of formula I. Mice receiving subcutaneous drug wereconcomitantly administered vehicle topically to both eyes to mimic thewetting of the ocular surface that occurs with a topical eye drop.Draining CLN were excised and prepared for flow cytometry analysis of Tcells (CD4, CD8), monocytes (CD11b), dendritic cells (CD11c) and MHC II.

One day of DS led to a significant increase in CD11b⁺ monocytes in CLNcompared to normal mice. None of the other cell populations studied weresignificantly altered by DS in draining CLN. None of the treatments hadan impact on either of the T cell populations or CD11b+ monocytes. WhileCD11c⁺ and CD11c⁺/MHC II⁺ cells tended to be increased by OS, albeit notsignificantly, topical treatment with the compound of formula I reducedboth, activated and non-activated, forms of DCs compared to the vehiclecontrol group. Neither systemic treatment with the compound of formula Inor topical treatment with dexamethasone phosphate produced the sameeffect. The results are presented in FIG. 4.

The fact that the compound of formula I inhibited antigen-presentingcell migration to the CLN and improved corneal staining in this murinemodel of dry eye only when administered topically, but not whenadministered systemically, is an intriguing finding. It suggests a localrather than systemic effect by the integrin α4 antagonist in thetreatment of DED. This local effect appears to be specific to theintegrin α4 antagonist GW559090 and differentiated from topical steroidtreatment (dexamethasone phosphate) in that topical dexamethasonephosphate ameliorated DED associated corneal staining, but not themigration of antigen-presenting cells to the draining lymph nodes. Itcan be appreciated by someone skilled in the art from thoseconsiderations that the therapeutic effect of integrin α4 antagonismemploys a unique and novel mechanism.

Example 6. PK Study in Rabbits

The potassium salt of GW559090, referred to herein as GW559090A, wasgiven to male New Zealand White rabbits by topical ocular administrationas a single bilateral drop (Study #1: n=2 rabbits/4 eyes per time point;Study #2: n=3 rabbits/6 eyes per time point). At designated times (0.5,1, 3, 6 hours post administration), ocular tissues were collected andprocessed for analysis. Concentrations of GW559090 were measured incornea and bulbar conjunctiva by liquid chromatography with tandem massspectrometry (LC-MS/MS).

Following a single topical administration of 40 μL (50 mg/mL), Cmaxvalues in bulbar conjunctiva were observed at 1 hour postdose (Tmax) forboth studies. Mean conjunctiva concentrations generally declined overtime and were quantifiable through 6 hours postdose. Following a singletopical administration of 40 μL (50 mg/mL), mean Cmax values in corneawere observed at 0.5 hours postdose (Tmax) for both studies. Mean corneaconcentrations generally declined over time and were quantifiablethrough 6 hours postdose. Mean cornea concentrations were higher thanmean conjunctiva concentrations at the 3 and 6 hour postdose timepoints. Conjunctival and corneal tissue concentrations of approximately358 ng/mL persisted up to 6 hours (FIG. 6). The conjunctival and cornealtissue concentrations were in excess of 3000 ng/mL at 30 minutes andlevels persisted above 1000 ng/mL in corneal tissue at 3 hours (FIG. 6).

Example 7. Topical Ocular Administration to New Zealand White Rabbitsfor 13 Weeks

GW559090A was used in this study. Doses and concentrations in thisExample are expressed in terms of the parent compound, GW559090.

GW559090A was given to New Zealand White Rabbits by topical ocularadministration for 13 weeks. Animals were dosed six or twelve times perday, with one hour between doses. Treatment groups were: (1) vehicle (12times a day), (2) 30 mg/mL/occasion given six times a day, (3) 50mg/mL/occasion given six times a day, or (4) 50 mg/mL/occasion, given 12times a day.

GW559090A was formulated as a stock solution in 25 mM phosphate bufferwith 0.5% sodium chloride to provide a 50 mg/mL stock solution, whichwas used for administration to Groups 3 or 4, or further diluted with 25mM phosphate buffer with 0.75% sodium chloride to provide a 30 mg/mLsolution for administration to Group 2. Control animals were treatedwith the vehicle for 50 mg/mL solution (25 mM phosphate buffer with 0.5%sodium chloride).

During the 13 week treatment period rabbits received daily topicalocular doses of GW559090A or vehicle at a constant dose volume of 0.06mL (given 6 or 12 times a day, with one hour between doses). The testarticle formulation or vehicle was carefully dropped into the partiallyeverted lower eyelid of the right eye from a suitable distance, toprevent contact of the positive displacement pipette with the eye. Thelower and upper lids were then gently held together for approximatelyfive seconds, to prevent loss of dose and aid even distribution acrossthe external ocular surface. The un-dosed eye was not manipulated at anytime and served as a within animal untreated control site.

Instillation of GW559090A for 13 weeks by topical ocular administrationat 30 or 50 mg/mL/occasion six times a day or 50 mg/mL/occasion, 12times a day, with one hour between doses, was well-tolerated and causedno ocular change or systemic toxicity. The No-Observed-Adverse-Effectlevel (NOAEL) was 50 mg/mL/occasion, 12 times a day, with one hourbetween doses. At this dose, based on Week 13 values, AUC0-1 (0-24)exposure was 19.6 (235) ng·h/mL for males and 35.5 (426) ng·h/mL forfemales; Cmax was 38.1 g/mL for males and 68.0 ng/mL for females.

The composite mean toxicokinetic parameters for GW559090 from male andfemale rabbits following ocular administration of GW559090A are shown inTable 2 and Table 3, respectively.

TABLE 2 Composite Mean Toxicokinetic Parameters for GW559090 from MaleRabbits Following Ocular Administration of GW559090A Male Dose ofGW559090 (mg/mL) Parameter Period 30^(a) 50^(a) 50^(b)AUC_(0.1)(AUC₀₋₂₄) Day 1 7.86 (47.2)  14.4 (86.4) 12.1 (145) (ng · h/mL)Week 4 9.23 (55.4) 17.9 (107) 11.6 (139) Week 13 9.10 (54.6) 17.6 (106)NA Week 14 NA NA 19.6 (235) C_(max) ^(c) Day 1 30.8 105 66.3 (ng/mL)Week 4 51.4 80.4 41.4 Week 13 11.8 32.2 NA Week 14 NA NA 38.1 T_(max)^(c) Day 1 0.083 0.083 0.083 (h) Week 4 0.083 0.083 0.083 Week 13 0.0830.083 NA Week 14 NA NA 0.5 ^(a)6 daily doses ^(b)12 daily doses^(c)After the first daily dose NA: Not Applicable

TABLE 3 Composite Mean Toxicokinetic Parameters for GW559090 from FemaleRabbits Following Ocular Administration of GW559090A Female Dose ofGW559090 (mg/mL) Parameter Period 30^(a) 50^(a) 50^(b) AUC₀₋₁(AUC₀₋₂₄)Day 1 4.19 (25.1) 11.0 (66.0)  7.30 (87.6) (ng · h/mL) Week 4 10.6(63.6) 12.6 (75.6) 13.1 (157) Week 13 9.83 (59.0) 15.7 (94.2) NA Week 14NA NA 35.5 (426) C_(max) ^(c) Day 1 16.0 67.8 30.6 (ng/mL) Week 4 32.444.4 56.6 Week 13 23.5 49.7 NA Week 14 NA NA 68.0 T_(max) ^(c) Day 10.083 0.083 0.083 (h) Week 4 0.083 0.083 0.083 Week 13 0.25 0.083 NAWeek 14 NA NA 0.083 ^(a)6 daily doses ^(b)12 daily doses ^(c)After thefirst daily dose NA: Not Applicable “AUC_(0.24)” refers to AUC₀₋₁multiplied by total number of daily doses to represent total dailyestimated AUC.

Example 8. Topical Ocular Administration to Beagle Dogs for 13 Weeks

GW559090A was used in this study. Doses and concentrations in thisExample are expressed in terms of the parent compound, GW559090.

GW559090A was given to Beagle Dogs by topical ocular administration for13 weeks. Animals were dosed six or twelve times per day, with one hourbetween doses. Treatment groups were: (1) vehicle (12 times a day), (2)30 mg/mLoccasion given six times a day, (3) 50 mg/mLioccasion given sixtimes a day, or (4) 50 mg/mLioccasion, given 12 times a day.

GW559090A was formulated as a stock solution in 25 mM phosphate bufferwith 0.5% sodium chloride to provide a 50 mg/mL stock solution, whichwas used for administration to Groups 3 or 4, or further diluted with 25mM phosphate buffer with 0.75% sodium chloride to provide a 30 mg/mLsolution for administration to Group 2.

During the 13 week treatment period dogs received daily topical oculardoses of GW559090 or vehicle (12 times a day) at a constant dose volumeof 0.06 mL (given 6 or 12 times a day, with one hour between doses). Thetest article formulation or vehicle was carefully dropped into theeverted lower eyelid of the left eye from a suitable distance, toprevent contact of the positive displacement pipette with the eye. Thelower and upper lids were then gently held together for approximatelyfive seconds, to prevent loss of dose and aid even distribution acrossthe external ocular surface. The un-dosed eye was not manipulated at anytime and served as a within animal untreated control site.

Instillation of the test article under the lower eyelid of the left eyeup to 12 times daily at concentrations up to 50 mg/mL waswell-tolerated. There were no findings considered to be attributable tolocal or systemic effects of the test article.

In conclusion, topical ocular administration of GW559090 to beagle dogsfor 13 weeks at 30 or 50 mg/mL/occasion six times daily or 50mg/mL/occasion 12 times daily, with one hour between doses, waswell-tolerated and caused no ocular change or systemic toxicity. TheNo-Observed-Adverse-Effect level (NOAEL) was therefore 50 mg/mL/occasiongiven 12 times a day. At this dose, based on Week 13 values males andfemales combined, AUC₀₋₁ exposure was 11.2 ng·h/mL (estimated AUC₀₋₂₄was 134 ng·h/mL); Cmax was 28.7 ng/mL.

A summary of toxicokinetic values for GW559090 from male and female dogsfollowing ocular administration of GW559090A are shown in Table 4 andTable 5, respectively.

TABLE 4 Toxicokinetic Parameters for GW559090 from Male Dogs FollowingOcular Administration of GW559090A Dose of GW559090 (mg/mL) 30^(b)50^(b) 50^(c) Parameter Period Male AUC₀₋₁(AUC₀₋₂₄) Day 1 3.82 (22.9)18.4 (110)  36.6 (439) (ng · h/mL) Week 4 2.27 (13.6) 8.74 (52.4) 11.8(142) Week 13 3.81 (22.9) 6.36 (38.2) 10.2 (122) C_(max) ^(a) Day 1 7.4152.8 69.7 (ng/mL) Week 4 5.30 24.2 23.8 Week 13 7.03 13.2 26.8 T_(max)^(a) Day 1 0.5 0.083 0.375 (h) Week 4 0.25 0.167 0.25 Week 13 0.5 0.250.25 ^(a)After the first daily dose ^(b)6 daily doses ^(c)12 daily dosesNA: Not Applicable

TABLE 5 Toxicokinetic Parameters for GW559090 from Female Dogs FollowingOcular Administration of GW559090A Dose of GW559090 (mg/mL) 30^(b)50^(b) 50^(c) Parameter Period Female AUC₀₋₁(AUC₀₋₂₄) Day 1 5.27 (31.6)8.79 (52.7) 12.8 (153) (ng · h/mL) Week 4 4.28 (25.7) 5.26 (31.6)  6.77(81.2) Week 13 6.05 (36.3) 6.64 (39.8) 12.1 (146) C_(max) ^(a) Day 112.2 17.0 21.8 (ng/mL) Week 4 9.34 11.4 14.5 Week 13 17.5 17.9 30.5T_(max) ^(a) Day 1 0.5 0.25 0.5 (h) Week 4 0.375 0.25 0.167 Week 13 0.250.167 0.25 ^(a)After the first daily dose ^(b)6 daily doses ^(c)12 dailydoses NA: Not Applicable

Example 9. A Randomized, Double-Masked, Placebo ControlledParallel-Group Design to Evaluate the Safety, Tolerability and Efficacyof GW559090 in Dry Eye Patients

This is a two-part study. Part 1 will be conducted at one or morecenters and is an open-label dose de-escalation tolerability trial ofGW559090 in healthy volunteers. Upon identification of a tolerable dose,the study will move to Part 2. Part 2 is a prospective, placebocontrolled, randomized, double-masked, parallel group, multi-centrestudy assessing the safety and efficacy of GWA/559090 for the treatmentof dry eye disease (DED).

Part 1 will have up to two treatment arms. Up to 10 healthy volunteerswill receive 50 mg/mL GW559090 TID in one eye for a period of 7 days. Ifthis dose is tolerated in 10 patients, the study will proceed to Part 2,and a lower dose will not be explored. If the 50 mg/mL is poorlytolerated, a second cohort of 10 healthy volunteers will receive 30mg/mL GW559090 TID in one eye for a period of 7 days. If this dose istolerated, the study will proceed to Part 2. Evaluation of tolerabilitywill be based upon review of physical findings and responses to thetolerability questionnaire. The study will be terminated if neither doseis tolerated. Maximum duration of participation in this phase of theprotocol for an individual patient will be 22 days (14 days screening, 7days GW559090, 1 day to follow-up).

In Part 2, approximately 90 subjects with moderate to severe DED will beentered into a placebo run-in period during which they will receive onedrop of placebo TID over 14 days. After the placebo run-in period, thefirst approximately 76 subjects who still meet inclusion criteria andhave demonstrated compliance with dosing in the run-in period will berandomized to one of two arms in 2:1 ratio (GW559090:placebo). Subjectswill be randomized to either the highest tolerated dose of GW559090(from Part 1) delivered three times a day, or a dose of placebo(vehicle) three times a day. Subjects will continue drug as assigned forup to 12 weeks. The maximum duration of participation of subjects inthis phase of the protocol will be 115 days (14 days screening, 14 daysplacebo run-in, 84 days treatment, and 3 days to follow-up).

Part 2 of the protocol is intended to establish proof of concept in adry eye population. Given that the efficacy observed in pre-clinicalmodels was non-inferior to dexamethasone, it is reasonable to expectthat meaningful effects will be seen on an objective physiologicalbiomarker (corneal staining) that directly reflects epithelial integrityin the eye. A double blind randomized approach is appropriate asmultiple end points are subject to reporting bias, by either theinvestigator or the subject. A placebo (vehicle) arm is included becauseprior studies have demonstrated pronounced placebo effects due in partto the lubricating effect of vehicle eye drops. A clinical resulttherefore cannot be assessed without accounting for this effect.

Example 10. Study to Evaluate the Safety, Tolerability and Efficacy ofGW559090 in Dry Eye Patients

In this study, approximately 200 subjects with moderate to severe DEDwill initially be screened. Approximately 120 subjects will berandomized to one of two arms in 1:1 ratio (GW559090:placebo). Subjectswill be randomized to either the highest tolerated dose of GW559090(e.g., 50 mg/mL) delivered three times a day, or a dose of placebo(vehicle) three times a day. The duration of this phase of the studywill be 98-101 days (14 days placebo run-in, 84 days treatment, and,optionally, 3 days to follow-up).

Upon completion of an interim analysis, additional subjects will then berandomized into a parallel dose-ranging study. If the interim analysisreveals a strong response to TID of the highest tolerated dose,approximately 200 subjects will be randomized to one of five groups:placebo TID; 3 mg/mL TID; 30 mg/mL TID; 50 mg/mL TID; or 50 mg/mL BID.If the interim analysis reveals a moderate response to TID of thehighest tolerated dose, approximately 140 subjects will be randomized toone of four groups: placebo TID; 3 mg/mL TID; 30 mg/mL TID; or 50 mg/mLTID.

REFERENCES

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Exemplary Embodiments

A1. A pharmaceutical composition comprising a compound of formula Iwhich is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof, and one or more pharmaceutically acceptableexcipients.

A2. The pharmaceutical composition of embodiment A1, wherein thecomposition is applied topically.

A3. The pharmaceutical composition of embodiment A1, wherein thecomposition is applied to the conjunctival sac or to the eyelid.

A4. The pharmaceutical composition of embodiment A1, wherein thecomposition is applied subconjunctivally, intracamerally,intravitreally, subtenon, subretinally, subchoroidally, orsuprachoroidally.

A5. The pharmaceutical composition of embodiments A2, A3 or A4, which isuseful for treating an ocular inflammatory condition.

A6. The pharmaceutical composition of any of the preceding embodiments,wherein the composition is applied in the form of an eye drop, spray ormist.

A7. The pharmaceutical composition of any of the preceding embodiments,wherein the composition is applied with an insert or other deliverydevice.

A8. A method for treatment of an ocular inflammatory condition in amammal/human in need thereof comprising administering to saidmammal/human a therapeutically effective amount of a compound of formulaI which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof, and one or more pharmaceutically acceptableexcipients.

A9. The method of embodiment A8, wherein the composition is appliedtopically.

A10. The method of embodiment A8, wherein the composition is applied tothe conjunctival sac or to the eyelid.

A11. The method of embodiment A8, wherein the composition is appliedsubconjunctivally, intracamerally, intravitreally, subtenon,subretinally, subchoroidally, or suprachoroidally.

A12. The method of any of the preceding embodiments, wherein thecomposition is applied in the form of an eye drop, spray or mist.

A13. The method of any of the preceding embodiments, wherein thecomposition is applied with an insert or other delivery device.

A14. A pharmaceutical composition comprising a compound of formula iwhich is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof, and one or more pharmaceutically acceptableexcipients, for use in the treatment of an ocular inflammatorycondition.

A15. A method for treating an ocular inflammatory condition in amammal/human in need thereof by blocking the migration ofantigen-presenting cells to the lymph nodes, which method comprisesadministering to said mammal/human a therapeutically effective amount ofcompound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof, and one or more pharmaceutically acceptableexcipients.

A16. A method for treatment of an ocular inflammatory condition in amammal/human in need thereof comprising administering to saidmammal/human a therapeutically effective amount of: (a) a pharmaceuticalcomposition comprising a compound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof; and (b) cyclosporin A, and one or morepharmaceutically acceptable excipients.

A17. A method for treatment of an ocular inflammatory condition in amammal/human in need thereof comprising administering to saidmammal/human a therapeutically effective amount of: (a) a pharmaceuticalcomposition comprising a compound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof; and (b) a topical steroid selected from the groupconsisting of dexamethasone base and phosphate, difluprednate,fluocinolone, fluorometholone base and acetate, loteprednol,prednisolone acetate and phosphate, rimexolone, and triamcinoloneacetonide, and one or more pharmaceutically acceptable excipients.

A18. A method for treatment of an ocular inflammatory condition in amammal/human in need thereof comprising administering to saidmammal/human a therapeutically effective amount of: (a) a pharmaceuticalcomposition comprising a compound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof; and (b) a non-steroidal anti-inflammatory drugselected from the group consisting of bromfenac, diclofenac,flurbiprofen, ketorolac, and nepafenac, and one or more pharmaceuticallyacceptable excipients.

A19. A method for treatment of an ocular inflammatory condition in amammal/human in need thereof comprising administering to saidmammal/human a therapeutically effective amount of: (a) a pharmaceuticalcomposition comprising a compound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof; and (b) an LFA-1 integrin antagonist, and one or morepharmaceutically acceptable excipients.

A20. The method of embodiment A19, wherein the LFA-1 integrin antagonistis lifitegrast.

A21. The method of embodiments A15, A16, A17, A18, A19, or A20, whereinthe composition is applied topically in the form of an eye drop, sprayor mist.

A22. Use of compound of formula I which is

or any pharmaceutically acceptable isomer, hydrate, anhydride, solvate,ester, salt form, free acid or base, prodrug, complex, conjugate, orpolymorph thereof, and one or more pharmaceutically acceptableexcipients, in the manufacture of a medicament for the treatment of anocular inflammatory condition.

A23. The pharmaceutical composition, method, or use of any of thepreceding embodiments, wherein the ocular inflammatory condition is dryeye disease, non-infectious uveitis (e.g., anterior, intermediate,posterior, or pan-uveitis), non-infectious conjunctivitis, iritis, orscleritis.

A24. The pharmaceutical composition, method, or use of any of thepreceding embodiments, wherein the ocular inflammatory condition is dryeye disease.

What is claimed is:
 1. A method of treatment, comprising: ocularlyadministering to a human subject in need of acute therapy for an ocularinflammatory condition: (a) a pharmaceutical composition comprising atherapeutically effective amount of a compound of formula I

 or a pharmaceutically acceptable salt, ester, hydrate, solvate,polymorph, optical isomer, racemate, diastereoisomer or enantiomerthereof, and (b) an effective amount of an additional therapeutic agent,wherein the additional therapeutic agent is (i) cyclosporin A or (ii)lifitegrast.
 2. The method of claim 1, wherein the ocular inflammatorycondition is selected from the group consisting of dry eye disease,noninfectious uveitis, non-infectious conjunctivitis, iritis, andscleritis.
 3. The method of claim 1, wherein the pharmaceuticalcomposition is administered with an individual dose of the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof in the range from about 0.01 mg to about 2 mg, ineach affected eye.
 4. The method of claim 1, wherein the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof is administered for a period of up to four weeks. 5.The method of claim 1, wherein the compound of formula I or thepharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof isadministered for a period of up to six weeks.
 6. The method of claim 1,wherein the compound of formula I or the pharmaceutically acceptablesalt, ester, hydrate, solvate, polymorph, optical isomer, racemate,diastereoisomer or enantiomer thereof is administered for a period of upto eight weeks.
 7. The method of claim 1, wherein the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof is administered for a period of up to ten weeks. 8.The method of claim 1, wherein the compound of formula I or thepharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof isadministered for a period of up to twelve weeks.
 9. The method of claim1, wherein the additional therapeutic agent is lifitegrast and isadministered in the range from about 0.01 mg to 10 mg, in each affectedeye.
 10. The method of claim 1, wherein the additional therapeutic agentis cyclosporine A and is administered in the range from about 0.001 mgto about 0.1 mg, in each affected eye.
 11. A method of treatment,comprising: ocularly administering to a human subject in need of acutetherapy for dry eye disease: (a) a pharmaceutical composition comprisinga therapeutically effective amount of a compound of formula I

 or a pharmaceutically acceptable salt, ester, hydrate, solvate,polymorph, optical isomer, racemate, diastereoisomer or enantiomerthereof, and (b) an effective amount of an additional therapeutic agent,wherein the additional therapeutic agent is (i) cyclosporin A or (ii)lifitegrast.
 12. The method of claim 11, wherein the pharmaceuticalcomposition is administered with an individual dose of the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof in the range from about 0.01 mg to about 2 mg, ineach affected eye.
 13. The method of claim 11, wherein the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof is administered for a period of up to four weeks. 14.The method of claim 11, wherein the compound of formula I or thepharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof isadministered for a period of up to six weeks.
 15. The method of claim11, wherein the compound of formula I or the pharmaceutically acceptablesalt, ester, hydrate, solvate, polymorph, optical isomer, racemate,diastereoisomer or enantiomer thereof is administered for a period of upto eight weeks.
 16. The method of claim 11, wherein the compound offormula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof is administered for a period of up to ten weeks. 17.The method of claim 11, wherein the compound of formula I or thepharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof isadministered for a period of up to twelve weeks.
 18. The method of claim11, wherein the additional therapeutic agent is lifitegrast and isadministered in the range from about 0.01 mg to 10 mg, in each affectedeye.
 19. The method of claim 11, wherein the additional therapeuticagent is cyclosporine A and is administered in the range from about0.001 mg to about 0.1 mg, in each affected eye.
 20. A method oftreatment, comprising, ocularly administering to a human subjectsuffering from dry eye disease: (a) a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of formula I

 or a pharmaceutically acceptable salt, ester, hydrate, solvate,polymorph, optical isomer, racemate, diastereoisomer or enantiomerthereof, and (b) an effective amount of an additional therapeutic agent,wherein the additional therapeutic agent is: (i) cyclosporin A and isadministered in the range from about 0.001 mg to about 0.1 mg, in eachaffected eye, or (ii) lifitegrast and is administered in the range fromabout 0.01 mg to 10 mg, in each affected eye; and wherein the compoundof formula I or the pharmaceutically acceptable salt, ester, hydrate,solvate, polymorph, optical isomer, racemate, diastereoisomer orenantiomer thereof is administered with an individual dose of thecompound in the range of from about 0.01 mg to about 2 mg, in eachaffected eye, for a period of up to twelve weeks.
 21. The method ofclaim 20, wherein the compound of formula I or the pharmaceuticallyacceptable salt, ester, hydrate, solvate, polymorph, optical isomer,racemate, diastereoisomer or enantiomer thereof is administered for aperiod of up to four weeks.
 22. The method of claim 20, wherein thecompound of formula I or the pharmaceutically acceptable salt, ester,hydrate, solvate, polymorph, optical isomer, racemate, diastereoisomeror enantiomer thereof is administered for a period of up to six weeks.23. The method of claim 20, wherein the compound of formula I or thepharmaceutically acceptable salt, ester, hydrate, solvate, polymorph,optical isomer, racemate, diastereoisomer or enantiomer thereof isadministered for a period of up to eight weeks.
 24. The method of claim20, wherein the compound of formula I or the pharmaceutically acceptablesalt, ester, hydrate, solvate, polymorph, optical isomer, racemate,diastereoisomer or enantiomer thereof is administered for a period of upto ten weeks.